Catalyzed reporter deposition (CARD) is a novel method of signal amplification which constitutes the subject matter of U.S. Pat. Nos. 5,731,158; 5,583,001 and 5,196,306. These patents, and all other patents referred to herein, are incorporated by reference. Assays employing this technique are also described in Bobrow et al., Journal of Immunological Methods, 125:279-285 (1989) and in Bobrow et al., Journal of Immunological Methods, 137-103-112 (1991).
The CARD method utilizes an analyte-dependent enzyme activation system (“ADEAS”) to catalyze the deposition of reporter or hapten groups, collectively referred to as “labels,” onto a receptor, the receptor being part of or added to a surface in contact with the components of the assay. These enzymatically deposited labels are detected directly or indirectly, resulting in signal amplification and improved detection limits. In the previously disclosed references, a peroxidase was the preferred enzyme. While peroxidase enzymes give reliable assay results, the kinetics of the peroxidase reaction are, in some instances, overly rapid. Such rapid kinetics can decrease the dynamic range of the assay, making it difficult to collect quantitative data over broad ranges. Hydrolytic enzymes, also referred to as hydrolases, generally have slower kinetics, and it is desirable to incorporate them in these assays.
In the CARD method, an enzyme reacts with a conjugate consisting of a labeled compound incorporated into an enzyme substrate. When the enzyme and the substrate react, a reactive intermediate, also termed an “activated conjugate,” is formed which binds covalently wherever receptor for the reactive intermediate is immobilized. Examples of such compounds which have been described for use with peroxidase enzymes include substituted phenols such as biotinyl-tyramide, fluorescein tyramide and p-hydroxycinnamoyl-containing substrates disclosed in U.S. Pat. No. 5,863,748, as well as the 4-(4-hydroxystyryl)pyridine substrates disclosed in U.S. Pat. No. 6,355,443.
In other analyses of the prior art, enzyme substrates have been used to generate products which become colored, fluorescent or chemiluminescent. These products either remain soluble or become insoluble and precipitate on a surface. The CARD method differs in that the reactive intermediates become covalently bound to the receptor, which may be part of, or separately, immobilized on a surface.
2-difluoromethylphenyl and p-hydroxymandelic acid compounds are well known for use as, for example, activity-based probes, Zhu et al., (2003) Tetrahedron Letters, 44, 2669-2672, Lo et al., (2002) J. Proteome Res., 1, 35-40; screening of antibody libraries, Cesaro-Tadic et al., (2003) Nature Biotechnology, 21, 679-685, Janda et al., (1997) Science 275, 945-948; and suicide substrates, Halazy et al., (1990) Bioorganic Chemistry 18, 330-344, Betley et al., (2002) Angew. Chem. Int. Ed. 41, 775-777. However, none of these references teaches the use of these compounds as substrates for detection and amplification in analyte-dependent assays such as immunoassays, nucleic acid hybridization assays, microarray assays, immunohistochemical applications, in-situ hybridization applications and the like.